Specific gene polymorphisms in breast cancer diagnosis, prevention and treatment

ABSTRACT

A method for detecting a predisposition to breast cancer in a subject is provided. The method includes detecting in a biological sample from the subject one or more polymorphisms in the sequence of CD44 gene. The presence of one or more polymorphisms in the sequence of CD44 gene indicates that the subject has a predisposition for developing breast cancer.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims is based on and claims priority to U.S.Provisional Application Ser. No. 61/191,079, filed Sep. 5, 2008, whichis incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under 5R01A/05373-06awarded by the National Institutes of Health. Therefore, the governmenthas certain rights in the invention.

BACKGROUND

Breast cancer is a leading cause of cancer-related deaths in women inthe United States and worldwide. Inherited genetic variations, or genepolymorphisms, are viewed as major factors in breast cancer prevalence.

A number of breast cancer susceptibility genes have been identified. Thetwo most important breast cancer susceptibility genes, BRCA1 and BRCA2,accounted for approximately 15% of breast cancer cases due to inheritedmutations or alterations. However, BRCA1 and BRCA2 mutations are presentin far less than 1% of the general population. In addition to BRCA1 andBRCA2, five other genes, TP53, PTEN, LKB1, ATM and CHEK2, are also wellestablished breast cancer susceptibility genes. The proportion of breastcancer in the general population explained by these gene mutations isvery small. It is estimated that all the currently known breast cancersusceptibility genes accounts for less than 25% of the familialaggregation of breast cancer. The evidence to date strongly suggeststhat the majority of the familial clustering of breast cancer isunexplained and other breast cancer susceptibility genes still remain tobe identified. The identification of new genes could make a major impactin risk prediction.

CD44 is a cell surface transmembrane glycoprotein, encoded by a singlegene. Transcripts for CD44 gene undergo complex alternative splicingthat results in many functionally distinct isoforms. Recent experimentaland clinical evidence shows that CD44 and its interaction withhyaluronan may regulate breast cancer cell proliferation, migration, andinvasion, as well as tumor-associated angiogenesis and are correlatedwith patient survival. Thus, CD44 polymorphisms play an important rolein breast cancer development.

SUMMARY

Objects and advantages of the invention will be set forth in part in thefollowing description, or may be obvious from the description, or may belearned through the practice of the invention.

In accordance with certain embodiments of the present disclosure, amethod for detecting a predisposition to breast cancer in a subject isprovided. The method comprises detecting in a biological sample from thesubject one or more polymorphisms in the sequence of CD44 gene. Thepresence of one or more polymorphisms in the sequence of CD44 geneindicates that the subject has a predisposition for developing breastcancer.

In still another embodiment of the present disclosure, a diagnostic kitfor identifying a predisposition to breast cancer in a subject isprovided. The kit comprises packaging material and means for amplifyingat least a region of a CD44 gene from a biological sample of a subject.

Other features and aspects of the present disclosure are discussed ingreater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure, including the best mode thereof,directed to one of ordinary skill in the art, is set forth moreparticularly in the remainder of the specification, which makesreference to the appended figures in which:

FIG. 1 is the sequence of a cloned PCR product from a PBMC sample ofbreast cancer patient (SEQ ID NO: 3).

FIG. 2 is a single nucleotide polymorphism analysis ofS703779.USU-1-M13R clone (polymorphic change in bold). FIG. 2 disclosesresidues 61-120 of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, in orderof appearance.

FIG. 3 is the CD44 polymorphism in the CD44 exon2 coding region from abreast cancer patient A (polymorphic change in CD44 exon2 codingsequence in bold). FIG. 3 discloses SEQ ID NO: 5, residues 61-180 of SEQID NO: 5 and SEQ ID NO: 6, respectively, in order of appearance.

FIG. 4 is the CD44 polymorphism in the CD44 exon2 coding region from abreast cancer patient B (polymorphic change in CD44 exon2 codingsequence in bold). FIG. 4 discloses SEQ ID NO: 7, residues 61-240 of SEQID NO: 7, and SEQ ID NO: 8, respectively, in order of appearance.

FIG. 5 is the CD44 polymorphism in the CD44 exon2 coding region from abreast cancer patient C (polymorphic change in CD44 exon2 codingsequence in bold), FIG. 5 discloses SEQ ID NO: 9, residues 1-240 of SEQID NO: 9, and SEQ ID NO: 10, respectively, in order of appearance.

FIG. 6 illustrates an association of CD44 polymorphisms and patient ageat breast cancer diagnosis.

Table 1 is a summary of unique CD44 polymorphism (Ex2+14 A>G) in breastcancer patients.

Table 2 is a comparison of unique CD44 polymorphism (Ex2+14 A>G) betweenCaucasian breast cancer patients and Caucasian normal donors.

Table 3 is a summary of additional CD44 polymorphisms in CD44 exon2coding sequence in breast cancer patients.

DETAILED DESCRIPTION

Reference now will be made in detail to various embodiments of thedisclosure, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the disclosure, notlimitation of the disclosure. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present disclosure without departing from the scope or spirit ofthe disclosure. For instance, features illustrated or described as partof one embodiment, can be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present disclosurecovers such modifications and variations as come within the scope of theappended claims and their equivalents.

Generally, the present disclosure provides a method of testing a subjectto determine if the subject has a predisposition for developing breastcancer.

The methods of the present disclosure provide that either healthysubjects and/or subjects at risk (after primary breast cancer) arescreened by obtaining various patient-derived materials such as tissuesamples or blood (normally blood, such as peripheral blood mononuclearcells, including lymphocytes or a monocytes), which is then examinedusing methods known to those in the art for the presence ofsingle-nucleotide polymorphism (also referred to herein generally aspolymorphisms). The tissue sample can include, but are not limited to,blood, mouth brush secretions, other secretions and other tissues.

The methods which are used to detect the presence of the polymorphismsinclude, but are not limited to methods known in the art for amplifyingor sequencing the one or more polymorphisms and detecting the one ormore polymorphisms or sequences thereof. Methods can include methodswhich require the use of primers, as further described herein. Themethods can include one or more of polymerase chain reaction, ligasechain reaction, restriction digestion, direct sequencing, nucleic acidamplification, microchips, hybridization or immunoassays. The methodsdescribed herein can further include a visualization method of thebreast including one or more of MRI, mammography, or ultrasonography onthe subject to detect breast cancer.

The present disclosure describes CD44 polymorphisms as a good marker inbreast cancer disease prediction and diagnosis. In particular, thepresent disclosure reveals patients with breast cancer have asignificantly increased frequency of CD44 Ex2+14 A>G polymorphism. Thepredisposition may be an inherited predisposition, as described furtherherein.

The present disclosure also provides a diagnostic kit for identifying apredisposition to breast cancer, including packaging material and one ormore different polynucleotides capable of amplifying at least a regionof a CD44 gene. In some embodiments of the present disclosure, theamplified region includes CD44 Ex2+14 A>G polymorphism. The kit may alsocontain instructions, e.g., instructions for using the kit to identify apredisposition to breast cancer, including instructions for performing avisualization method of the breast. In addition, the kit can allow forthe ability to sequence at least a region of a CD44 gene from abiological sample of a user of the diagnostic kit. For instance, the kitcan include packaging to send a biological sample to be sequenced by aDNA sequencer, or another method, as further described above.

(1) Unique CD44 Polymorphism in Breast Cancer Patients:

Since CD44 polymorphisms are present in breast cancer patients and areassociated with breast cancer development, the direct nucleotidesequencing strategy was used to analyze CD44 polymorphisms in breastcancer patients. A unique polymorphism in the boundary region betweenCD44 exon2 and its upstream intron was identified in breast cancerpatients, which is located in the upstream intron of CD44 exon2 (FIGS. 1and 2). The polymorphic change is A→G, and its position is +14 from CD44exon2. This unique CD44 polymorphism was designated as the Ex2+14 A>Gpolymorphism. The Caucasian breast cancer patients had a significantlyhigher frequency of this unique polymorphism than Asian breast cancerpatients (Table 1), which may be associated with the fact that whitewomen have a higher incidence and death rate from breast cancer thanwomen of other racial and ethnic groups. As compared with the Caucasiannormal donors, the Caucasian patients with breast cancer have asignificantly increased frequency of CD44 Ex2+14 A>G polymorphism (Table2). These results indicated that the identified CD44 polymorphism playsan important role in breast cancer development.

(2) Polymorphisms in CD44 Exon2 Coding Sequence in Breast CancerPatients:

CD44 is a major cell surface receptor for hyaluronan (HA), a componentof the extracellular matrix. Many of the functions of CD44 in celladhesion and activation can be attributed to CD44 binding. Exon2 iscritical for CD44 binding to HA. Thus, there is interest inunderstanding the polymorphisms in the CD44 exon2 coding region andtheir role in breast cancer development. The direct nucleotidesequencing analysis indicated that different polymorphic changes in theCD44 exon2 coding region were identified in different breast cancerpatients (FIGS. 3, 4 and 5). Therefore, except the unique CD44 Ex2+14A>G polymorphism in the boundary region between CD44 exon2 and itsupstream intron, the additional polymorphisms in the CD44 exon2 codingsequence are also present in breast cancer patients. Thus, the breastcancer patients were divided into two groups: (1) Group I patients: thepatients contained only unique CD44 Ex2+14 A>G polymorphism and (2)Group II patients: the patients contained the additional CD44polymorphisms in CD44 exon2 coding sequence.

Caucasian breast cancer patients had a significantly higher frequency ofthe polymorphisms in the CD44 exon2 coding sequence than Asian breastcancer patients (Table 3). No polymorphisms in the CD44 exon2 codingsequence have been identified in elderly Caucasian normal donors. Theincidence and death rates from breast cancer are lower among women ofother racial and ethnic groups than among white and African-Americanwomen, indicating that the polymorphisms in the CD44 exon2 codingsequence play an important role in breast cancer development.

(3) Relationship Between CD44 Polymorphisms and Breast CancerDevelopment:

As shown above, Caucasian breast cancer patients have a significantlyincreased frequency of the unique CD44 Ex2+14 A>G polymorphism in theboundary region between CD44 exon2 and its upstream intron thanCaucasian normal donors (Table 2), indicating that this unique CD44polymorphism plays an important role in breast cancer development. Theadditional polymorphisms in CD44 exon2 coding sequence have beenidentified only in breast cancer patients (FIGS. 3, 4 and 5; Table 3),but not in elderly Caucasian normal donors. Therefore, understanding theclinical significance of CD44 polymorphisms in breast cancer developmentis important. Mann-Whitney U test analysis shows that the patientscontaining the additional CD44 polymorphisms in CD44 exon2 codingsequence had breast cancer disease at earlier stage, which isstatistically significant (FIG. 6), indicating that CD44 polymorphismmay play role in breast cancer disease development.

The following examples are meant to illustrate the disclosure describedherein and are not intended to limit the scope of this disclosure.

EXAMPLES Technology for CD44 Polymorphism Analysis

-   Step 1: Obtain normal cells such as peripheral blood mononuclear    cells (PBMC) from human subjects.-   Step 2: Isolate genomic DNA samples from normal cell samples.-   Step 3: Use polymerase chain reaction (PCR) method to amplify CD44    exon2 region sequences including the CD44 exon2 sequence and the    boundary sequences between exon2 and upstream intron and between    exon2 and downstream intron from genomic DNA samples in the presence    of the forward primer (5′-GGAGTCTGTCCTAAACTGA ACTTATTACTG-3′ (SEQ ID    NO: 1)) and the reverse primer (5′-AGCTTTCCCCAGTGGTCGGGTGC    TGGTCTC-3′ (SEQ ID NO: 2)).-   Step 4: Clone PCR products of CD44 exon2 region sequences using a    cloning vector such as pCR2.1 TOPO from Invitrogen.-   Step 5: Transform the cloned PCR products into bacterial competent    cells such as TOP10 cells from Invitrogen.-   Step 6: Isolate plasmid DNA samples containing the cloned CD44 exon2    region sequences using plasmid DNA preparation kit such as QIAprep    spin miniprep kit from QIAGEN.-   Step 7: Sequence the plasmid DNA samples containing the cloned CD44    exon2 region sequences using automated DNA sequencer.-   Step 8: Analyze the polymorphic changes in CD44 exon2 region    including CD44 exon2 coding sequence and the adjacent intron    sequences.-   Step 9: Use the information about CD44 polymorphisms to prevent,    predict and diagnose breast cancer.

It has been well documented that the two most important breast cancersusceptibility genes, BRCA1 and BRCA2, accounted for approximately 15%of breast cancer cases due to inherited mutations or alterations.However, BRCA1 and BRCA2 mutations are present in far less than 1% ofthe general population. It is estimated that all the currently knownbreast cancer susceptibility genes accounts for less than 25% of thefamilial aggregation of breast cancer. Our data demonstrated that CD44gene polymorphisms/variations may account for 80% of breast cancercases. Thus, CD44 polymorphisms will be a good marker in breast cancerdisease prediction and diagnosis. The technique of CD44 polymorphismanalysis used in breast cancer patients is feasible and reliable so thatCD44 polymorphism analysis has a great potential in clinical prevention,diagnosis and treatment for breast cancer patients.

Early diagnosis of cancer diseases is a big challenge in effectivecancer treatment. Genetic screen of CD44 polymorphisms in breast cancerpatients will provide a novel platform for cancer prevention and earlycancer disease diagnosis, which will be of benefit to the effectiveclinical treatment of breast cancer disease.

In the interest of brevity and conciseness, any ranges of values setforth in this specification are to be construed as written descriptionsupport for claims reciting any sub-ranges having endpoints which arewhole number values within the specified range in question. By way of ahypothetical illustrative example, a disclosure in this specification ofa range of 1-5 shall be considered to support claims to any of thefollowing sub-ranges: 1-4; 1-3; 1-2; 2-5; 2-4; 2-3; 3-5; 3-4; and 4-5.

These and other modifications and variations to the present disclosurecan be practiced by those of ordinary skill in the art, withoutdeparting from the spirit and scope of the present disclosure, which ismore particularly set forth in the appended claims. In addition, itshould be understood that aspects of the various embodiments can beinterchanged both in whole or in part. Furthermore, those of ordinaryskill in the art will appreciate that the foregoing description is byway of example only, and is not intended to limit the disclosure so asfurther described in such appended claims.

TABLE 1 Female breast cancer patients Caucasian Asian Number of patientscontaining unique CD44 33 12 polymorphism Number of patients withoutunique CD44 8 11 polymorphism Total number of patients analyzed 41 23Percentage of patients containing unique CD44 80 52 polymorphism (%)Note: Statistical analysis indicated that Caucasian female breast cancerpatients had a significant higher percentage of the patients containingunique CD44 Ex2 + 14 A > G polymorphism than Asian female breast cancerpatients (Odds ratio, 3.78; 95% confidence intervals, 1.23-11.65; p <0.05).

TABLE 2 Caucasian population Cancer patient Normal donor Number ofsubjects containing unique 33 4 CD44 polymorphism Number of subjectswithout unique 8 5 CD44 polymorphism Total number of subjects analyzed41 9 Percentage of subjects containing 80 44 unique CD44 polymorphism(%) Note: Statistical analysis indicated that Caucasian female breastcancer patients had a significant higher percentage of the patientscontaining unique CD44 Ex2 + 14 A > G polymorphism than Caucasian normaldonors (Odds ratio, 5.16; 95% confidence intervals, 1.12-23.69; p <0.05).

TABLE 3 Female breast cancer patients Caucasian Asian Number of patientscontaining unique 16 7 CD44 polymorphism only Number of patientscontaining 17 2 additional CD44 polymorphisms Total number of patientsanalyzed 33 9 Percentage of patients containing 52 22 additional CD44polymorphisms (%) Note: Statistical analysis indicated that Caucasianfemale breast cancer patients had a significant higher percentage of thepatients containing additional CD44 polymorphisms than Asian femalebreast cancer patients (Odds ratio, 3.72; 95% confidence intervals,0.67-20.63).

1. A method for detecting a predisposition to breast cancer in asubject, comprising: obtaining a biological sample from a subject;isolating genomic DNA from the biological sample; amplifying CD44 exon2region sequences including the CD44 exon2 sequence and boundarysequences between exon2 and upstream intron and between exon2 anddownstream intron from the genomic DNA in the presence of a forwardprimer SEQ ID NO: 1 and a reverse primer SEQ ID NO: 2 to formamplification products; and detecting in the amplification products oneor more polymorphisms, wherein the presence of one or more polymorphismsin the sequence of CD44 exon2 gene indicates that the subject has apredisposition for developing breast cancer.
 2. The method as in claim1, wherein the subject is a human.
 3. The method as in claim 1, whereinthe biological sample comprises blood.
 4. The method as in claim 1,wherein the biological sample comprises peripheral blood mononuclearcells.
 5. The method as in claim 1, further comprising performing avisualization method of a breast of the subject comprising one or moreof MRI, mammography, or ultrasonography to detect breast cancer.
 6. Themethod as in claim 1, wherein the predisposition is an inheritedpredisposition.
 7. The method as in claim 1, wherein the one or morepolymorphisms in the sequence of CD44 gene comprises Ex2+14 A>G.
 8. Themethod as in claim 1, further comprising cloning the amplificationproducts to form cloned PCR products.
 9. The method as in claim 8,further comprising transforming the cloned PCR products into bacterialcompetent cells to develop plasmid DNA samples containing the cloned PCRproducts.
 10. The method as in claim 9, further comprising isolating theplasmid DNA samples containing the cloned PCR products.
 11. The methodas in claim 10, further comprising sequencing the plasmid DNA samples.12. The method as in claim 11, further comprising analyzing thepolymorphisms and the adjacent intron sequences.